1. Field of the Invention
The present invention relates to 3-phosphoglycerate dehydrogenase variants whose inhibition by L-serine is reduced, and to genes encoding them.
2. The Prior Art
The twenty natural, protein-forming amino acids are nowadays in the main prepared by fermenting microorganisms. In this connection, use is made of the fact that microorganisms possess appropriate biosynthetic pathways for synthesizing the natural amino acids.
However, in wild-type strains, these biosynthetic pathways are subject to strict control which ensures that the amino acids are only produced to satisfy the endogenous requirement of the cell. An example of an important control mechanism in many biosyntheses is the phenomenon of feedback inhibition (or end product inhibition). In feedback inhibition, it is usually the enzyme in a biosynthetic pathway, which catalyzes the initial enzyme reaction of this biosynthetic pathway, which is inhibited by the end product of the biosynthetic pathway. The inhibition is usually effected by the end product binding allosterically to the enzyme and bringing about a conformational change which converts the enzyme into an inactive state. This thereby ensures that, when the end product accrues in the cell, further synthesis is stopped by the introductory step being inhibited.
It is therefore only possible to produce metabolic products (such as amino acids) efficiently on an industrial scale when the restrictions resulting from the feedback inhibition of a metabolic pathway can be abolished. This will thereby make available microorganisms which, as compared with wild-type organisms, exhibit a drastic increase in their ability to produce the desired metabolic product.
The phosphoglycerate family of amino acids is defined by the fact that these amino acids are derived biosynthetically from 3-phosphoglyceric acid. In this case, the natural path of the metabolism leads initially to L-serine by way of the intermediates 3-phosphohydroxypyruvate and 3-phospho-L-serine. L-serine can be subsequently converted into glycine or else, by way of O-acetylserine, into L-cysteine. L-tryptophan is also to be included in this group since it is likewise derived from the biosynthesis of L-serine. In the same way, unnatural amino acids which are prepared using the method described in US 2002/0039767 A1 are also to be assigned to the phosphoglycerate family.
Compounds which are derived from C1 metabolism are likewise dependent on the biosynthesis of the amino acids of the phosphoglycerate family. This is due to the fact that, when L-serine is converted into glycine, tetrahydrofolate acts as the C1 group acceptor and the loaded tetrahydrofolate is involved, as the central methyl group donor in C1 metabolism, in many biosyntheses (e.g. L-methionine, nucleotides, pantothenic acid, etc.). According to the invention, compounds which are derived from C1 metabolism are consequently preferably compounds whose biosynthesis depends on a C1 group transfer by way of tetrahydrofolic acid.
The initial step in the biosynthesis of amino acids belonging to the phosphoglycerate family is the oxidation of D-3-phosphoglyceric acid to 3-phosphohydroxypyruvate and is catalyzed by the enzyme 3-phosphoglycerate dehydrogenase (PGD) [EC 1.1.1.95]. NAD+, which is converted into NADH/H+, serves as the acceptor for the reducing equivalents which are formed in the reaction.
PGD enzymes are known from a very wide variety of organisms (e.g. Rattus norvegicus, Arabidopsis thaliana, Escherichia coli, Bacillus subtilis). The better characterized microbial representatives of these enzymes are subject to feedback inhibition by L-serine.
At the amino acid sequence level, the microbial PGD enzymes are very similar to each other in the N-terminal moiety (amino acids 1-340 in the case of the E. coli sequence) whereas the C-terminal moieties only exhibit slight similarities. However, it is precisely in this C-terminal moiety that the regulatory domain which is responsible for the serine inhibition is located (Peters-Wendisch et al., 2002, Appl. Microbiol. Biotechnol. 60:437-441).
The PGD which is best characterized is that from Escherichia coli. The enzyme has been investigated biochemically in detail (Dubrow & Pizer, 1977, J. Biol. Chem. 252, 1527-1538) and is subject to allosteric feedback inhibition by L-serine, with the inhibitor constant Ki being 5 μM.
This feedback inhibition stands in the way of efficiently producing amino acids belonging to the phosphoglycerate family and has therefore already been the target for molecular biological approaches.
Thus, the document EP0620853A described variants of Escherichia coli PGD which are less susceptible to inhibition by serine and which exhibit a modification in the C-terminal 25% of the wild-type PGD (i.e. amino acids 307-410), preferably a modification in the region of the last 50 residues (i.e. amino acids 361-410). The mutants which are described were obtained by linker mutagenesis, i.e. by simply making use of restriction cleavage sites which are present in the E. coli serA gene and inserting oligonucleotide linkers of 8-14 base pairs in length.
However, such linker mutageneses usually give rise to problems since incorporating, or deleting, several residues very greatly alters the structure of the protein and in this way has a negative influence on the overall activity or stability of the protein. In fact, most of the mutants described in EP0620853A have an activity which is scarcely detectable.
Mutageneses which achieve the goal of decreasing the susceptibility of PGD to inhibition by serine have also been performed on the serA gene in coryneform microorganisms:
Peters-Wendisch et al. (2002, Appl. Microbiol. Biotechnol. 60:437-441) describe C-terminal deletions of the Corynebacterium glutamicum PGD. In this case, too, the deletions lead to great loss of enzyme activity in some instances.
The application EP0943687A2 describes a replacement of the glutamic acid residue at position 325 in the Brevibacterium flavum PGD. In an alignment formed using the GAP algorithm of the GCG (GCG Wisconsin Package, Genetics Computer Group (GCG) Madison, Wis.) program, this residue is already located, with reference to the Escherichia coli PGD, in the variable C-terminal moiety of the protein and correlates with the asparagine residue 364 in the Escherichia coli protein. Since this modification is located in the variable C-terminal moiety of PGD, it is not possible to draw any conclusions with regard to the Escherichia coli protein.